Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the proposed mechanism of action of Yoda1 on PIEZO1 (left) and close-up view of the THU8–9 interface with previously proposed residues lining putative Yoda1 binding sites highlighted in sphere representation. (B) , time course of Ca 2+ -influx (F/F0) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) assessed by GCamp8 imaging together with comparison of maximum responses (right). (C) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) stretch-evoked currents by 30µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using Mann-Whitney test (PIEZO1: CTL = 4.02, N=14 vs Yoda1 = 1.38, N=15, P=0.0000131) and Student’s t-test(P1.A2094W: CTL = 8.35, N=10 vs Yoda1 = 1.19, N=10, P=0.0033), pressure-response curves (i.e. peak current amplitude at indicated pressure normalized to maximal response, bottom right) and comparison of P 50 values in the absence and presence of Yoda1 using Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N=14 vs Yoda1 = -16.9 ± 6.65 mmHg, N=13, P=0.006) and Mann-Whitney test (P1.A2094W: CTL = -43.7 ± 10.04 mmHg, N=10 vs Yoda1 = -27.4 ± 6.7 mmHg, N=10, P=0.0005). (D) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) poking-evoked currents in whole-cell recordings by 30µM Yoda1. Example traces evoked by incrementing (Δ 800nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using Mann-Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N=14 vs Yoda1 = 41.1 ± 21.6, N=12, P=0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N=18 vs Yoda1 = 8.3 ± 2.7, N=20, P=0.0405), displacement-response curves (i.e. peak current amplitude vs. indicated stimulus magnitude; bottom, right) and comparison of mechanical activation thresholds using Mann-Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N=14 vs Yoda1 = 2.8 ± 1.4 µm, N=12, P=0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N=18 vs Yoda1 = 3.8 ± 1.1 µm, N=20, P=0.0069).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Binding Assay, Expressing, Imaging, Comparison, MANN-WHITNEY, Activation Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of THU8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. (B) , Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket-2 and pocket-3 in the flat conformation. (C) , AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the overall strategy to resolve Yoda1 induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. (B) Confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). (C) Time-course of the average ± s.e.m. (from N=3-4 independent experiments) of the maximal Yoda1 (50µM) effect on the normalized fluorescence (F/F0) for PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom). (D-F) In-plane projections of representative trimers examples of PIEZO1 ( D ), A2094W ( E ) and V1714A_1715A ( F ) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50µM) (left). Comparison of the mean ± s.e.m. interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 ( D , N=93 and 110), A2094W ( E , N=61 and 64) and V1714A_1715A ( F , N=87 and 59), with unpaired t-test (right).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Expressing, Cryo-EM Sample Prep, Fluorescence, Comparison

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: 19 F NMR spectra of RBCs loaded with 5FBAPTA and treated with Yoda1 in the presence of Ca 2+ , at 37 °C. The peak from free 5FBAPTA inside the RBCs (initially 4 mmol [L RBC] −1 ) is highlighted in pink; yellow highlights the peak from the extracellular 5FBAPTA-calcium complex; green, the peak from the intracellular 5FBAPTA-calcium complex; and blue, the peak from the intracellular protein-5FBAPTA-calcium complex. The sample was 0.5 mL RBCs ( Ht = 0.73) in 154 mM NaCl and 10 mM d -glucose. The spectra were recorded every 10 min with NMR settings as for Fig. . The superimposed red spectrum at 7 min is from the 6th spectrum of a 1 h time course recorded with the RBCs in the presence of 2.0 µL 1 M CaCl 2 (corresponding to 4.0 mM Ca 2+ concentration averaged over the sample). Then, Yoda1 was added as 1.0 µL of 14 mM in DMSO; this value combined with the knowledge of the Ht value gave a concentration of 38 µmol [L RBC] −1 . Left inset: combined integral of the peak from 5FBAPTA-Ca (green plus yellow). Right inset: integral of intracellular free 5FBAPTA (pink). The fitted equations and their parameter values were (left and right, respectively): 1.06 ± 0.07 + (0.011 ± 0.003) t − (0.00005 ± 0.0003) t 2 , and 2.81 ± 0.09 − (0.010 ± 0.004) t − (0.00005 ± 0.0004) t 2 .

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: 19 F NMR spectra showing A23187 (and Yoda1 in comparison) stimulated uptake of Ca 2+ in RBCs loaded with 5FBAPTA (4 mmol [L RBC] −1 ), at 37 °C. Pink highlights the resonance corresponding to intracellular free 5FBAPTA that was assigned the chemical shift δ = 0.0 ppm; and the green highlights the peak from the calcium complex that was centred at ~ 5.8 ppm. The sample of 0.5 mL RBCs ( Ht = 0.62) was constituted in 154 mM NaCl, 10 mM glucose. ( a ) The RBCs had been loaded with 4 mM 5FBAPTA as described in Methods. Then, for ( b , c ), added with brisk mixing (by five-fold rapid inversion and re-inversion of the NMR tube) were: 5 µL 1 M CaCl 2 making the concentration 10 mM averaged over the volume of the sample; and 0.5 µL 20 mM A23187 in DMSO giving a concentration of 32 µmol (L RBC) −1 . For ( d ) 5 µL 1 M CaCl 2 and 1.0 µL 14 mM Yoda1 in DMSO giving a concentration of 45 µmol (L RBC) −1 were mixed into the 0.5 mL susupension. The time indicated on the right of each spectrum was the mid-point of spectral accumulation after a 2 min lag between mixing the sample and starting FID accumulation. NMR settings were as for Fig. except the time per spectrum was 10 min 47 s.

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Comparison, Concentration Assay

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: Graphical representation of an RBC under strain in a stretched gel (see below); it is loaded with the Ca 2+- sensing chelator 5FBAPTA that yields separate 19 F NMR signals from the free and Ca 2+ -complexed forms. Ca 2+ enters via the mechanosensitive cation channel Piezo1 that can be activated (+ symbol) by the small-molecule compound Yoda1. Ca 2+ entry into the RBC can also be mediated by the Ca 2+ -selective ionophore A23187. [1,6- 13 C] d -glucose enters the cell via the glucose transporter GLUT1; it was used in conjunction with 13 C NMR spectroscopy to measure glycolytic flux under various experimental conditions. The model of the distorted RBC is based on Cartesian translation in Mathematica with the shape defined by the parametric equations given in .

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Structural Proteomics